Fig 2. mDia1/3-dependent cortical F-actin meshwork in primary cultured Sertoli cells.

(A) TIRF 3D-N-STORM imaging of the actin filaments in WT primary cultured Sertoli cells. The images on the right side are magnified images of the corresponding white boxed areas of the lower magnification. Color bar indicates the z dimension (−300 nm to 300 nm). Scale bars, 5 μm (left) and 500 nm (right). (B) Quantification of the ratio of F-actin meshwork occupancy per cell area in WT or mDia1/3 DKO primary cultured Sertoli cells. Results of eight cells for each genotype from two independent experiments. Data are represented as mean ± SEM. ***P < 0.001 (Student t test). (C) Representative images of TIRF 3D-N-STORM imaging of the actin filaments in control WT primary cultured Sertoli cells or Sertoli cells treated with 30 μM SMIFH2 or 50 μM CK-666 for 1 h. Two SMIFH2-treated Sertoli cells with different extents of impaired cortical F-actin meshwork phenotype are shown. The images on the right side are magnified images of the corresponding white boxed areas of the lower magnification. Scale bars, 5 μm (left) and 500 nm (right). (D) Quantification of the ratio of F-actin meshwork occupancy per cell area in control, 50 μM CK-666–treated, or 30 μM SMIFH2-treated primary cultured Sertoli cells. Results of nine cells for control, eight cells for CK-666–treated, and 15 cells for SMIFH2-treated primary cultured Sertoli cells from two independent experiments. *P < 0.05, ***P < 0.001 (P = 0.017 for control versus CK-666–treated cells, P < 0.001 for control versus SMIFH2-treated cells, and P < 0.001 for CK-666–treated versus SMIFH2-treated cells; one-way ANOVA with post hoc test). DKO, double knockout; F-actin, filamentous actin; mDia1/3, mammalian diaphanous homolog1/3; SMIFH2, small molecule inhibitor of formin homology 2 domain; TIRF, total internal reflection; WT, wild-type; 3D-N-STORM, three dimensional-N-stochastic optical reconstruction microscopy.